Oligomers and polymers of cyclic imino carboxylic acids

ABSTRACT

Disclosed are cyclic imino oligomers and polymers comprised of subunits of the formula:                    
     Also disclosed are combinatorial libraries and arrays of the cyclic imino compounds.

Priority is hereby claimed to provisional application Serial No.60/138,972, filed Jun. 14, 1999, the content of which is incorporatedherein by reference.

This invention was made with United States government support awarded bythe National Institutes of Health: NIH GM56414. The United States hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention is directed to unnatural polypeptide-likemolecules which are oligomers or polymers of constrained iminocarboxylic acids, methods of generating combinatorial libraries usingthese residues, and combinatorial libraries formed thereby.

DESCRIPTION OF THE PRIOR ART

Chemists have long sought to extrapolate the power of biologicalcatalysis and recognition to synthetic systems. These efforts havefocused largely on low molecular weight catalysts and receptors. Mostbiological systems, however, rely almost exclusively on large polymerssuch as proteins and RNA to perform complex chemical functions.

Proteins and RNA are unique in their ability to adopt compact,well-ordered conformations. These two biopolymers are unique alsobecause they can perform complex chemical operations (e.g., catalysis,highly selective recognition, etc.). Folding is linked to function inboth proteins and RNA because the creation of an “active site” requiresproper positioning of reactive groups. Consequently, there has been along-felt need to identify synthetic polymer backbones which displaydiscrete and predictable folding propensities (hereinafter referred toas “foldamers”) to mimic natural biological systems. Such backbones willprovide molecular “tools” to probe the functionality of large-moleculeinteractions (e.g. protein-protein and protein-RNA interactions).

Much work on β-amino acids and peptides synthesized therefrom has beenperformed by a group led by Dieter Seebach in Zurich, Switzerland. See,for example, Seebach et al. (1996) Helv. Chim. Acta. 79:913-941; andSeebach et al. (1996) Helv. Chim. Acta. 79:2043-2066. In the first ofthese two papers Seebach et al. describe the synthesis andcharacterization of a β-hexapeptide, namely(H-β-HVal-β-HAla-β-HLeu)₂—OH. Interestingly, this paper specificallynotes that prior art reports on the structure of β-peptides have beencontradictory and “partially controversial.” In the second paper,Seebach et al. explore the secondary structure of the above-notedβ-hexapeptide and the effects of residue variation on the secondarystructure.

Dado and Gellman (1994) J. Am. Chem. Soc. 116:1054-1062 describeintramolecular hydrogen bonding in derivatives of β-alanine and γ-aminobutyric acid. This paper postulates that β-peptides will fold in mannerssimilar to α-amino acid polymers if intramolecular hydrogen bondingbetween nearest neighbor amide groups on the polymer backbone is notfavored.

Suhara et al. (1996) Tetrahedron Lett. 37(10): 1575-1578 report apolysaccharide analog of a β-peptide in which D-glycocylaminederivatives are linked to each other via a C-1 β-carboxylate and a C-2α-amino group. This class of compounds has been given the trivial name“carbopeptoids.”

Regarding methods to generate combinatorial libraries, several recentreviews are available. See, for instance, Ellman (1996) Acc. Chem. Res.29:132-143 and Lam et al. (1997) Chem. Rev. 97:411-448.

SUMMARY OF THE INVENTION

The present invention is drawn to a genus of oligomers and polymers ofconformationally-restricted imino carboxylic acids. The preferredoligomers and polymers of the invention strongly favor a discretesecondary structure (although this is not a requirement of theinvention). These stable secondary structures include helices analogousto the well-known poly(proline) II helical structure seen in α-aminoacids.

More specifically, the invention is directed to compounds of theformula: comprising formula:

X—{A}_(n)—Y

wherein n is an integer greater than 1; and

each A, independent of every other A, is selected from the groupconsisting of:

wherein R¹, R², and R⁵ are independently selected from the groupconsisting of hydrogen, linear or branched C₁-C₆-alkyl, alkenyl, oralkynyl; mono-or di-C₁-C₆ alkylamino, mono- or bicyclic aryl, mono- orbicyclic heteroaryl having up to 5 heteroatoms selected from N, O, andS; mono- or bicyclic aryl-C₁-C₆-alkyl, mono- or bicyclicheteroaryl-C₁-C₆-alkyl, —(CH₂)₁₋₆—OR³, —(CH₂)₁₋₆—SR³,—(CH₂)₁₋₆—S(═O)—CH₂—R³, —(CH₂)₁₋₆—S(═O)₂—CH₂—R³, —(CH₂)₁₋₆—NR³R³,—(CH₂)₁₋₆—NHC(═O)R³, —(CH₂)₁₋₆—NHS(═O)₂—CH₂—R³, —(CH₂)₁₋₆—O—(CH₂)₂₋₆—R⁴,—(CH₂)₁₋₆—S—(CH₂)₂₋₆—R⁴, —(CH₂)₁₋₆—S(═O)—(CH₂)₂₋₆—R⁴,—(CH₂)₁₋₆—S(═O)₂—(CH₂)₂₋₆—R⁴, —(CH₂)₁₋₆—S(═O)₂—(CH₂)₂₋₆—R⁴,—(CH₂)₁₋₆—NH—(CH₂)₂₋₆—R⁴, —(CH₂)₁₋₆—N—{(CH₂)₂₋₆—R⁴}₂,—(CH₂)₁₋₆—NHC(═O)—(CH₂)₂₋₆—R⁴, and —(CH₂)₁₋₆—NHS(═O)₂—(CH₂)₂₋₆—R⁴;wherein

R³ is independently selected from the group consisting of hydrogen,C₁-C₆-alkyl, alkenyl, or alkynyl; mono- or bicyclic aryl, mono- orbicyclic heteraryl having up to 5 heteroatoms selected from N, O, and S;mono- or bicyclic aryl-C₁-C₆-alkyl, mono- or bicyclicheteroaryl-C₁-C₆-alkyl; and

R⁴ is selected from the group consisting of hydroxy, C₁-C₆-alkyloxy,aryloxy, heteroaryloxy, thio, C₁-C₆-alkylthio, C₁-C₆-alkylsulfinyl,C₁-C₆-alkylsulfonyl, arylthio, arylsulfinyl, arylsulfonyl,heteroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, amino, mono- ordi-C₁-C₆-alkylamino, mono- or diarylamino, mono- or diheteroarylamino,N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino,N-aryl-N-heteroarylamino, aryl-C₁-C₆-alkylamino, carboxylic acid,carboxamide, mono- or di-C₁-C₆-alkylcarboxamide, mono- ordiarylcarboxamide, mono- or diheteroarylcarboxamide,N-alkyl-N-arylcarboxamide, N-alkyl-N-heteroarylcarboxamide,N-aryl-N-heteroarylcarboxamide, sulfonic acid, sulfonamide, mono- ordi-C₁-C₆-alkylsulfonamide, mono- or diarylsulfonamide, mono- ordiheteroarylsulfonamide, N-alkyl-N-arylsulfonamide,N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide, urea;mono- di- or tri-substituted urea, wherein the subsitutent(s) isselected from the group consisting of C₁-C₆-alkyl, aryl, heteroaryl;O-alkylurethane, O-arylurethane, and O-heteroarylurethane;

R⁶ is selected from the group consisting of hydrogen, linear or branchedC₁-C₆-alkyl, alkenyl, or alkynyl; mono-or di-C₁-C₆ alkylamino, mono- orbicyclic aryl, mono- or bicyclic heteroaryl having up to 5 heteroatomsselected from N, O, and S; mono- or bicyclic aryl-C₁-C₆-alkyl, mono- orbicyclic heteroaryl-C₁-C₆-alkyl, —S(═O)₂—(CH₂)₁₋₆—R³, —C(═O)R³,—S(═O)₂—(CH₂)₂₋₆R⁴, and —C(═O)—(CH₂₎ ₁₋₆—R⁴;

wherein R³ and R⁴ are as defined above;

R⁷ and R⁸ are selected from the group listed above for R¹, R² and R⁵,and are further selected from the group consisting of hydroxy,C₁-C₆-alkyloxy, aryloxy, heteroaryloxy, thio, C₁-C₆-alkylthio,C₁-C₆-alkylsulfinyl, C₁-C₆-alkylsulfonyl, arylthio, arylsulfinyl,arylsulfonyl, heteroarylthio, heteroarylsulfinyl, heteroarylsulfonyl,amino, mono- or di-C₁-C₆-alkylamino, mono- or diarylamino, mono- ordiheteroarylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino,N-aryl-N-heteroarylamino, aryl-C₁-C₆-alkylamino, carboxylic acid,carboxamide, mono- or di-C₁-C₆-alkylcarboxamide, mono- ordiarylcarboxamide, mono- or diheteroarylcarboxamide,N-alkyl-N-arylcarboxamide, N-alkyl-N-heteroarylcarboxamide,N-aryl-N-heteroarylcarboxamide, sulfonic acid, sulfonamide, mono- ordi-C₁-C₆-alkylsulfonamide, mono- or diarylsulfonamide, mono- ordiheteroarylsulfonamide, N-alkyl-N-arylsulfonamide,N-alkyl-N-heteroarylsulfonamide, N-aryl-N-heteroarylsulfonamide, urea;mono- di- or tri-substituted urea, wherein the subsitutent(s) isselected from the group consisting of C₁-C₆-alkyl, aryl, heteroaryl;O-alkylurethane, O-arylurethane, and O-heteroarylurethane

one of X or Y is hydrogen or an amino-terminal capping group (such asformyl, acetyl, tBoc, Fmoc, etc.);

the other of X or Y is hydroxy or a carboxy-terminal capping group (suchas NH2, NH(alkyl), N(alkyl)₂, etc.);

and salts thereof.

As noted above, each “A” substituent is selected independently from oneanother. Consequently, the invention explicitly encompasses bothhomo-oligomers and polymers, as well as hetero-oligomers and polymers.

Encompassed within the invention are protected forms of the abovecompounds in which reactive carboxy and amino subtituents are protectedby selectively removable (including orthogonally removable) moieties.All substituents used as protecting groups in synthetic organicchemistry are encompassed within the definition. Expressly includedwithin this definition, without limitation, are carbamate-formingprotecting groups such as Boc, Fmoc, Cbz, and the like, andamide-forming protecting groups such as acetyl and the like. Suchprotecting groups are well known and widely used by those skilled in theart of peptide chemistry.

All stereochemical configurations (single enantiomers, singlediastereomers, mixtures thereof, and racemates thereof) of the compoundsdescribed above are encompassed within the scope of the invention. Inthe preferred embodiment, all of the residues share the same absoluteconfiguration (either R or S) about the asymmetric ring carbon in theposition a to the exocyclic carbonyl carbon.

The invention is further directed to a method for preparing acombinatorial library of the subject compounds, the method comprising atleast two successive iterations of first covalently linking a first {A}subunit via its C terminus to a plurality of separable solid substrates,the first subunit selected from the group recited above for “A,” andthen randomly dividing the plurality of substrates into at least twosub-groups and deprotecting the first subunits attached to the at leasttwo sub-groups. Then in separate and independent reactions, covalentlylinking to the first subunit of each of the at least two sub-groups asecond subunit independently selected from the above-listed group of {A}residues.

The invention is further drawn to a combinatorial library of oligomersand/or polymers comprising a plurality of different compounds asdescribed above, each compound covalently linked to a solid support, thecombinatorial library produced by the process described immediatelyabove.

Another embodiment of the invention is drawn to an array comprising aplurality of compounds as described above at selected, known locationson a substrate or in discrete solutions, wherein each of the compoundsis substantially pure within each of the selected known locations andhas a composition which is different from other polypeptides disposed atother selected and known locations on the substrate.

The primary advantage and utility of the present invention is that itallows the construction of synthetic peptides having high conformationalstability. These synthetic polyamides have utility in investigating thebiological interactions involving biopolymers. The stable secondarystructure of the present compounds allows them to mimic natural proteinsecondary structure, thereby allowing targeted disruption oflarge-molecule interactions (e.g., protein-protein interactions.)

It is also expected that the compounds of the present invention willreadily cross biological membranes due to their lower polarity ascompared to natural peptides. This is expected based upon the knownability of proline oligomers to cross biological membranes. Because thebackbone of the subject oligomers and polymers is linked by tertiaryamide bonds, the compounds lack acidic amide protons on the backbone.Additionally, because the compounds are unnatural, they are expected toresist enzymatic cleavage. Therefore, the subject oligomers have utilityas probes to investigate the ability of non-natural betapeptides tocross biological membranes.

As a natural consequence, the invention is further drawn to the use ofthese synthetic polyamides as base molecules from which to synthesizelarge libraries of novel compounds utilizing the techniques ofcombinatorial chemistry. In addition to varying the primary sequence ofthe residues, the ring positions of these compounds can be substitutedwith a wide variety of substituents, such as those described above forR¹ and R². The main advantage here is that substituents placed on thebackbone rings do not interfere with the ability of the compounds toadopt a regular secondary structure. Consequently, the subject compoundscan be utilized to construct vast libraries having differentsubstituents, but all of which share a stabilized secondary structure.This utility is highly desirable because, as a general principal,chemical structure is responsible for chemical activity. By providing ameans for constructing large libraries or arrays of the subjectcompounds, their structure-activity relationships can be cogentlyinvestigated by rational design of libraries or arrays containingsystematically altered permutations of the oligomers disclosed herein.

Other aims, objects, and advantages of the invention will appear morefully from a complete reading of the following Detailed Description ofthe Invention and the attached claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the CD spectra (in methanol) of nipecotic acid oligomersfrom the dimer to the hexamer.

FIG. 2 depicts CD spectra (in methanol) for an oligomeric series(monomer to hexamer) of pyrrolidine-3-carboxylic acid oligomers.

FIG. 3 depicts CD spectra (in methanol) for an oligomeric series(monomer to hexamer) of cis-5 methoxymethyl-3-pyrrolidine carboxylicacid (“cis-5-MOM-PCA”).

FIG. 4 depicts CD spectra (in methanol) comparing pentamers of the PCA,Nip, and cis-5-MOM-PCA compounds.

FIG. 5 is a schematic of the “split-pool” method of combinatorialchemistry.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions:

The following abbreviations are used throughout the specification andclaims. Unless specifically defined to the contrary, all other termshave their standard accepted meanings. All of the following compoundscan be purchased commercially from Aldrich Chemical Company, Milwaukee,Wis., USA, as well as other national and international suppliers:

“alkyl”=C₁-C₆ straight or branched alkyl

“Bn”=benzyl

“BnBr”=benzyl bromide

“Boc”=tert-butoxycarbonyl

“BopCl”=bis(2-oxo-3-oxazolidinyl)phosphinic chloride

“cis-5-MOM-PCA”=cis-5 methoxymethyl-3-pyrrolidine carboxylic acid

“Cbz”=carbobenzyloxy

“CSA”=(1S)-(+)-10-camphorsulfonic acid

“DIEA”=diisopropyl ethyl amine

“DMAP”=N,N-dimethylaminopyridine

“DMF”=N,N-dimethylformamide

“EDCI”=N,N-dimethylaminopropyl-3-ethylcarbodiimide

“FAB MS”=fast atom bombardment mass spectrometry

“MALDI-TOF MS”=matrix-assisted laser desorption ionization,time-of-flight mass spectrometry

“Nip”=nipecotic acid

“PCA”=pyrrolidine carboxylic acid

“PiCA”=piperazine carboxylic acid

“TCMP”=trans-3-carboxy-4-methylpiperidine

“TUF”=tetrahydrofuran

“Ts-Cl”=p-toluenesulfonyl chloride

Chemistry:

General. Melting points are uncorrected. CH₂Cl₂ was freshly distilledfrom CaH₂ under N₂. DMF was distilled under reduced pressure fromninhydrin and stored over 4 Å molecular sieves. Triethylamine wasdistilled from CaH₂ before use. Other solvents and reagents were used asobtained from commercial suppliers. For BOC removal, 4 M HCl in dioxanefrom was used. Column chromatography was carried out by using low airpressure (typically 6 psi) with 230-400 mesh silica gel 60. Routine¹H-NMR spectra were obtained on a Bruker AC-300 and are referenced toresidual protonated NMR solvent. Routine ¹³C-NMR spectra were obtainedon a Bruker AC-300 and are referenced to the NMR solvent. Highresolution electron impact mass spectroscopy was performed on a KratosMS-80RFA spectrometer with DS55/DS90.

Far UV Circular Dichroism (CD). Data were obtained on a Jasco J-715instrument at 20° C. In all CD plots contained herein, the mean residueellipticity is presented on the vertical axis. Presenting the meanresidue ellipticity is a standard practice in peptide chemistry whereinthe intensity of each CD spectrum is normalized for the number of amidechromophores in the peptide backbone. Consequently, when the intensityof the minimum (ca. 208 nm) peak characteristic of secondary structureformation increases with increasing chain length, this change representsan increase in the population of the secondary structure, rather thansimply an increase in the number of chromophores present in eachmolecule.

General Experimental Procedure A. Peptide Couplings Using Bop-Cl as theCoupling Reagent. Boc-Xxx-OBn (1.0 eq.) was dissolved in 4 N HCl/dioxane(2.5 eq.). The solution was stirred for 2 h, the solvent was removedunder a stream of N₂, and the residue was dried under vacuum to give awhite solid (Xxx-OBn.HCl). This material was dissolved in methylenechloride (0.1 M). Boc-Xxx-OH (1.0 eq.) was added and the reactionmixture was cooled to 0° C. BopCl (1.0 eq.) was added, followed by DEEA(2.0 eq.). The reaction mixture was stirred for 48 h at 5° C. in thecold room. The reaction mixture was removed from the cold room andpoured into a solution of diethyl ether (3×reaction volume) and H₂O(2×reaction volume). The organic layer was isolated and washed withsaturated KHSO₄, saturated NaHCO₃, and brine. The organic layer wasdried over MgSO₄ and concentrated. The crude product was then purifiedby column chromatography.

General Experimental Procedure B. Peptide Couplings Using Bop-Cl as theCoupling Reagent. Boc-Xxx-OBn (1.0 eq.) was dissolved in 4 N HCl/dioxane(2.5 eq.). The solution was stirred for 2 h, the solvent was removedunder a stream of N₂, and the residue was dried under vacuum to give awhite solid (Xxx-OBn.HCl). This material was dissolved in methylenechloride (0.2 M). Boc-Xxx-OH (1.0 eq.) was added and the reactionmixture was cooled to 0° C. BopCl (1.0 eq.) was added, followed by DEA(2.5 eq.). The reaction mixture was stirred for 48 h at roomtemperature. The reaction mixture was poured into a solution of diethylether (3×reaction volume) and H₂O (2×reaction volume). The organic layerwas isolated and washed with saturated KHSO4, saturated NaHCO₃, andbrine. The organic layer was dried over MgSO₄ and concentrated. Thecrude product was then purified by column chromatography.

Nipecotic Acid Oligomers

1. Synthesis of the Protected Monomer

Boc-(S)-Nip-OEt is the building block for the synthesis of nipecoticacid oligomers. The protected monomer was synthesized in three stepsbeginning with a resolution via co-crystallization with CSA. The aminogroup was then protected as the tert-butyl carbamate, and the carboxylgroup was protected as the ethyl ester.

(S)-Nip.(S)-CSA 1.1. (1S)-(+)-10-Camphorsulfonic acid (11.62 g, 0.05mol) was added to a stirred solution of racemic nipecotic acid (6.46 g,0.05 mol) in acetone (100 mL). The solution was heated to reflux, andH₂O (15 mL) was added until all solids dissolved. The solution wascooled to room temperature and allowed to stir overnight. Theprecipitate that formed was isolated by filtration and recrystallizedthree times with acetone/H₂O (6/1, v/v) to afford 1.99 g (11% yield) ofthe desired product as a white solid: m.p. 221-223° C.; {a}_(D)+25.3° (c1.0, MeOH).

Boc-(S)-Nip-OH 1.2. (S)-Nip.(S)-CSA (1.90 g, 5.3 mmol) was dissolved inmethanol (12 mL). Triethylamine (2.2 mL, 15.8 mmol) and di-tert-butyldicarbonate (1.38 g, 6.3 mmol) were added and the solution was stirredat 50° C. for 12 h. The solution was then concentrated, and the residuewas dissolved in H₂O. The aqueous solution was washed with diethylether, and the organic layer was discarded. The aqueous layer wasacidified to pH 3 with 1 M HCl and extracted with CH₂Cl₂. The organiclayer was dried over MgSO₄ and concentrated to afford 1.24 g(quantitative yield) of the desired product as a white solid: m.p.166-168° C.; FAB-MS m/z (M+Na⁺) calcd for C₁₁,H₁₉NO₄Na252.3, obsd 252.5.

Boc-(S)-Nip-OBn 1.3. Boc-(S)-Nip-OH (0.70 g, 3.1 mmol) was dissolved inN,N-dimethylformamide (DMF) (14 mL). Cs₂CO₃ (1.0 g, 3.1 I mmol) andbenzyl bromide (0.41 mL, 3.5 mmol) were added, and the solution wasstirred at room temperature for 24 h. The solution was thenconcentrated, and the residue was dissolved in H₂O. The aqueous solutionwas then extracted with CH₂Cl₂. The organic solution was dried overMgSO₄ and concentrated to give an oil. The crude product was purified bycolumn chromatography eluting with ethyl acetate/hexanes (1/3, v/v) toafford 0.81 g (82% yield) of the desired product as a white solid.

2. Oligomer Synthesis

Oligomers of nipecotic acid were synthesized in a stepwise fashion usingstandard coupling procedures:

The reaction scheme is as follows:

Boc-(S)-Nip-N(Me)₂1.4. Via general procedure A, HCl.N(Me)₂ (0.29 g, 3.5mmol) was coupled with Boc-(S)-Nip-OH (0.4 g, 1.7 mmol). After workup,the crude product was purified by column chromatography eluting withethyl acetate/hexanes (1/1, v/v) to afford 0.29 g (65% yield) of thedesired product as a colorless oil; FAB-MS m/z (M+Na⁺) calcd forC₁₁H₁₉NO₄Na⁺ 279.3, obsd 279.1.

Boc-{(S)-Nip}₂-OBn 1.5. Via general procedure A, Boc (S)-Nip-OBn (0.80g, 2.5 mmol) was Boc-deprotected and coupled with Boc-(S)-Nip-OH (0.64g, 2.5 mmol). After workup, the crude product was purified by columnchromatography eluting with ethyl acetate/hexanes (1/1, v/v) to afford0.69 g (64% yield) of the desired product as a colorless oil;MALDI-TOF-MS m/z (M+Na⁺) calcd for C₂₄H₃₄N₂O₅Na⁺ 453.5, obsd 453.3.

Boc-{(S)-Nip}₃-OBn 1.6. Via general procedure A, Boc {(S)-Nip-}₂OBn(0.37 g, 0.85 mmol) was Boc-deprotected and coupled with Boc-(S)-Nip-OH(0.19 g, 0.85 mmol). After workup, the crude product was purified bycolumn chromatography eluting with ethyl acetate/methanol (10/1, v/v) toafford 0.22 g (49% yield) of the desired product as a white foam;MALDI-TOF-MS m/z (M+Na⁺) calcd for C₃₀H₄₃N₃O₆Na⁺ 564.3, obsd 564.3.

Boc-{(S)-Nip}₄-OBn 1.7. Via general procedure A, Boc {(S)-Nip-}₂OBn(0.29 g, 0.62 mmol) was Boc-deprotected and coupled withBoc-{(S)-Nip}₂—OH (0.29 g, 0.85 mmol). After workup, the crude productwas purified by column chromatography eluting with ethylacetate/methanol (10/1, v/v) to afford 0.27 g (68% yield) of the desiredproduct as a white foam; MADI-TOF-MS m/z (M+Na⁺) calcd for C₃₆H₅₂N₄O₇Na⁺675.4, obsd 675.4.

Boc-{(S)-Nip}₅-OBn 1.8. Via general procedure A, Boc-(S)-Nip-OBn (88.4mg, 0.28 mmol) was Boc-deprotected and coupled with Boc-{(S)-Nip}₄—OH(0.14 g, 0.28 mmol). After workup, the crude product was purified bycolumn chromatography eluting with ethyl acetate/methanol (10/1, v/v) toafford 0.11 g (52% yield) of the desired product as a white foam;MALDI-TOF-MS m/z (M+Na⁺) calcd for C₄₂H₆₁N₅O₈Na⁺ 786.4, obsd 786.5.

Boc-{(S)-Nip}₆-OBn 1.9. Via general procedure A, Boc {(S)-Nip-}₂OBn(0.46 g, 1.1 mmol) was Boc-deprotected and coupled withBoc-{(S)-Nip}₄—OH (0.16 g, 0.3 mmol). After workup, the crude productwas purified by column chromatography eluting with ethylacetate/methanol (10/1, v/v) to afford 0.12 g (45% yield) of the desiredproduct as a white foam; MALDI-TOF-MS m/z (M+Na⁺) calcd forC₄₈H₇₀N₆O₉Na⁺ 897.5, obsd 897.6.

The CD spectra of the nipecotic acid oligomers from the dimer to thehexamer are shown in FIG. 1. The data have been normalized for b-peptideconcentration and number of amide groups. The CD spectrum of the trimerhas a different maxima and minima than the monomer and dimer. Thissuggests that the trimer is adopting a different secondary structure. Asthe oligomer is lengthened to tetramer and pentamer, the intensity ofthe spectra increase, which suggests that the secondary structure isbecoming more stable. The hexamer has the same intensity as thepentamer, suggesting that adding additional residues does not increasethe stability of the Nip oligomer. The isodichroic point at 218 nmindicates that only one distinct secondary structure is populated.

Circular dichroism data for 0.5 mM Nip pentamer in isopropanol as afunction of temperature indicate that the Nip oligomers are thermallystable, and that only at 75 ° C. does the stability of the oligomerdecrease (data not shown). Circular dichroism data for 0.5 mM Niphexamer (25° C.) protected in methanol and deprotected in H₂O, pH=7.6suggest that the same secondary structure is adopted in H₂O, with therebeing a small decrease in the stability of the structure (data notshown).

The synthesis of this monomer is an extension of that given in Klein etal. (1997), Bio. & Med. Chem. Let. 7:1773.

3-Hydroxy-(R)-Pyrrolidine. trans-4-Hydroxy-L-proline (13.11 g, 0.1 mol)was added to cyclohexanol (65 mL), followed by the addition of2-cyclohexene-1-one (0.65 mL). The reaction mixture was heated at 180°C. until all solids were dissolved. The solution was cooled to roomtemperature and concentrated by vacuum rotary evaporation. The crudeproduct was carried on to the next synthetic step without furtherpurification.

3-Hydroxy-Cbz-(R)-Pyrrolidine 2.1. 3-Hydroxy-(R)-pyrrolidine (8.71 g,0.1 mol) was dissolved in CH₂Cl₂ (260 mL) and cooled to 0° C.Triethylamine (33.5 mL, 0.24 mol) and benzyl chloroformate (14.9 mL,0.11 mol) were added, and the resulting solution was stirred for 2 h at0° C. The solution was gradually warmed to room temperature and allowedto stir overnight. The solution was washed with 1 M HCl, saturatedNaHCO₃, and brine. The organic solution was dried over MgSO₄ andconcentrated. The crude product was purified by column chromatographyeluting with ethyl acetate to afford 13.7 g (62% yield, 2 steps) of thedesired product as a purple oil.

3-Tosyl-Cbz-(R)-Pyrrolidine 2.2. 3-Hydroxy-Cbz-(R)-pyrrolidine (13.7 g,0.06 mol) was dissolved in CH₂Cl₂ (250 mL) and cooled to 0° C.p-Toluenesulfonyl chloride (14.16 g, 0.07 mol), and triethylamine (20.7mL, 0.15 mol) were added and the resulting solution was stirred for 4 hat 0° C. The solution was washed with 1 M HCl, saturated NaHCO₃, andbrine. The organic solution was dried over MgSO₄ and concentrated. Thecrude product was purified by column chromatography eluting with ethylacetate/hexanes (3/1, v/v) to afford 20.4 g (88% yield) of the desiredproduct as an oil.

3-Cyano-Cbz-(S)-Pyrrolidine 2.3. 3-Tosyl-Cbz-(R)-pyrrolidine (20.4 g,0.05 mol) was dissolved in DMSO (54 mL), followed by the addition of KCN(5.3 g, 0.08 mol). The reaction mixture was stirred for 5 h at 80° C.The solution was cooled to room temperature and brine/H₂O (90 mL) (1/1,v/v) was added. The aqueous solution was extracted with ethyl acetate.The organic extracts were dried over MgSO₄, and concentrated. The crudeproduct was purified by column chromatography eluting with ethylacetate/hexanes (1/1, v/v) to afford 8.13 g (65% yield) of the desiredproduct as an oil.

Cbz-(S)-PCA-OMe 2.4. 3-Cyano-Cbz-(S)-pyrrolidine (8.13 g, 35.3 mmol) wasdissolved in methanol (35 mL), followed by the addition of concentratedHCl (35 mL). The solution was stirred for 3 days at room temperature.The solution was neutralized by NaHCO₃. The methanol was removed and thesolution was diluted with H₂O (100 mL). The aqueous solution wasextracted with CH₂Cl₂. The organic extracts were dried over MgSO₄ andconcentrated. The crude product was purified by column chromatographyeluting with ethyl acetate/hexanes (1/1, v/v) to afford 5.26 g (57%yield) of the desired product as a colorless oil.

Boc-(S)-PCA-OMe 2.5. Cbz-(S)-PCA-OMe (5.26 g, 20.0 mmol) was dissolvedin methanol (0.1 M), 10% Pd/C (12% vol), and Boc₂O (5.67 g, 25.9 mmol)were added, and the solution was shaken on a Parr appartus for 12 hunder psi of H₂. The solution was filtered through a plug of glass wool,and the filtrate was concentrated. The crude product was purified bycolumn chromatography eluting with ethyl acetate/hexanes (1/1, v/v) toafford 3.79 g (83% yield) of the desired product as an colorless oil.

Boc-(S)-PCA-OH 2.6. Boc-(S)-PCA-OMe (2.52 g, 11.0 mmol) was dissolved inmethanol (155 mL) and H₂O (54 mL) and the solution was cooled to 0° C.LiOH.H₂O (4.6 g, 0.11 mol) was added, followed by H₂O₂ (6.23 mL, 0.05mol) and the solution was stirred for 15 h in the cold room at 5° C.While still cold, Na₂SO₃ (21 g, 0.17 mol) in H₂O (93 mL) was added. Themethanol was removed and the solution was bought to pH 2 with 1 M HCl.The aqueous solution was extracted with methylene chloride. The organicextracts were dried over MgSO₄ and concentrated to afford 2.36 g (88%yield) of the desired product as a white solid.

Boc-(S)-PCA-OBn 2.7. Boc-(S)-PCA-OH (1.7g, 4.9 mmol) was disolved in DMF(50 mL). Cs₂CO₃ (1.62 g, 4.9 mmol) and benzol bromide (0.63 mL, 5.2mmol) were added, and the solution was stirred for 24 h at roomtemperature. The solution was then concentrated, and the residue wasdissolved in H2O. The aqueous solution was then extracted with ethylacetate. The organic solution was dried over MgSO₄ and concentrated. Thecrude product was purified by column chromatography eluting with ethylacetate/hexanes (1/3, v/v) to afford 1.52 g (90% yield) of the desiredproduct as a white solid.

Boc-(S)-PCA-N(Me)₂ 2.8. Via general procedure A, HCl.N(Me)₂ (31.7 mg,0.5 mmol) was coupled with Boc-(S)-PCA-OH (93.5 mg, 0.4 mmol). Afterworkup, the crude product was purified by column chromatography elutingwith ethyl acetate/hexanes (1/1, v/v) to afford 39.1 mg (37% yield) ofthe desired product as a colorless oil.

Boc-{(S)-PCA}₂-OBn 2.9. Via general procedure A, Boc-(S)-PCA-OBn (0.46g, 1.5 mmol) was Boc-deprotected and coupled with Boc-(S)-PCA-OH (0.32g, 1.5 mmol). After workup, the crude product was purified by columnchromatography eluting with ethyl acetate/hexanes (3/1, v/v) to afford0.28 g (47% yield) of the desired product as an colorless oil.

Boc-{(S)-PCA}₃-OBn 2.10. Via general procedure A, Boc-{(S)-PCA}₂-OBn (90mg, 0.2 mmol) was Boc-deprotected and coupled with Boc-(S)-PCA-OH (48mg, 0.2 mmol). After workup, the crude product was purified by columnchromatography eluting with ethyl acetate/methanol (20/1, v/v) to afford29.8 mg (27% yield) of the desired product as an colorless oil.

Boc-{(S)-PCA}₄-OBn 2.11. Via general procedure A, Boc-{(S)-PCA}₂-OBn(0.21 g, 0.5 mmol) was Boc-deprotected and coupled withBoc-{(S)-PCA}₂—OH (0.16 g, 0.5 mmol). After workup, the crude productwas purified by column chromatography eluting with ethylacetate/methanol (20/1, v/v) to afford 67.9 mg (27% yield) of thedesired product as a white foam; MALDI-TOF-MS m/z (M⁺) calcd forC₃₂H₄₄N₄O₇Na 620.724, obsd 620.5.

Boc-{(S)-PCA}₅-OBn 2.12. Via general procedure A, Boc-{(S)-PCA}₃-OBn(0.10 g, 0.2 mmol) was Boc-deprotected and coupled with Boc-(S)-PCA-OH(36 mg, 0.2 mmol). After workup, the crude product was purified bycolumn chromatography eluting with methylene chloride/methanol (20/1,v/v) to afford 16.3 mg (14% yield) of the desired product as a clear,glassy solid; MALDI-TOF-MS m/z (M⁺) calcd for C₃₇H₅₁N₅O₈Na 716.841, obsd716.5.

Boc-{(S)-PCA}₆-OBn 2.13. Via general procedure A, Boc-{(S)-PCA}₄-OBn(0.10 g, 0.2 mmol) was Boc-deprotected and coupled withBoc-{(S)-PCA}₂—OH (52 mg, 0.2 mmol). After workup, the crude product waspurified by column chromatography eluting with methylenechloride/methanol (20/1, v/v) to afford 7.0 mg (6% yield) of the desiredproduct as a clear, glassy solid; MALDI-TOF-MS m/z (M⁺) calcd forC₄₂H₅₈N₆O₉Na 813.957, obsd 813.5.

Referring now to FIG. 2, circular dichroism data for PCA oligomers inmethanol (25° C.) suggest that the monomer 2.8 and dimer 2.9 adopt arandom conformation. The CD spectra of the trimer 2.10 is different,which suggests that the oligomer is starting to adopt a distinctsecondary structure. The CD spectra of the tetramer 2.11, pentamer 2.12,and hexamer 2.13 have a more intense signal than the trimer. Thissuggests that the secondary structure is more stable in going to thetetramer, but going to any higher oligomer, pentamer or hexamer, doesnot increase the structure's stability. An isodichroic point at 232 nmindicates that only one distinct secondary structure is being populated.

Circular dichroism data for 0.5 mM PCA pentamer 2.12 in isopropanol as afunction of temperature, 25° C., 50° C., and 75° C., indicate that theoligomers are thermally stable. An increase in the temperature onlymodestly decreases the intensity of the oligomers (data not shown).Circular dichroism data for 0.5 mM PCA tetramer (25° C.) protected inmethanol and deprotected in H₂O, pH=7.6, suggest that the PCA tetrameradopts the same secondary structure in H₂O; however, the stability ofthe oligomer has been decreased (data not shown).

Substituted Pyrrolidine-3-Carboxylic Acids

The following reaction illustrates the synthesis of apyrrolidine-3-carboxylic acid derivative bearing an amino group at the4-position:

Starting material: Blake, J. et al J. Am. Chem. Soc. 1964, 86, 5293.

The final product, compound 58, can be oligomerized in the same fashionas the other monomers described herein.

Compound 52: Compound 51 (2.0 g) and NaBH₃CN (0.54 g) were dissolved inmethanol (40 ml), 1N HCl (aqueous) was added dropwise to maintain pH3-4. After 15-20 minutes, pH change slowed. The mixture was stirred foran additional 1.0 hour, while 1N HCl was added occasionally to keep pH3-4. Water (100 ml) was added. The mixture was extracted diethyl ether(3×150 ml). The extracts were washedwith 1N NaHCO3 (100 ml) and dilutebrine (100 ml), dried over MgSO₄, and concentrated to give a colorlessoil (1.9 g) in 95% yield. The product was used directly without furtherpurification.

Compound 53: Compound 52 (1.9 g) and Ph₃P (2.8 g) were dissolved intoluene (anhydrous, 30 ml) under nitrogen. A solution of diethylazodicarboxylate (1.5 ml) in toluene (10 ml) was subsequenely introducedvia syringe over 15 minutes. The reaction mixture was stirred undernitrogen at room temperature for 12 hours. The toluene was removed underreduced pressure. The residue was purified by column chromatography withethyl acetate/hexane (3/7, v/v) as eluent to afford a colorless oil (1.6g) in 91% yield.

Compound 54: Compound 53 (1.0 g) and R-(+)-α-methylbenzylamine (1.1 ml)were mixed with water (15 ml). The mixture was stirred at 55° C. for 67hours. The mixture was taken up in diethyl ether (300 ml), and theaqueous layer was separated. The ether solution was washed with water(3×50 ml), dried over MgSO₄, and concentrated to give a slight yellowoil. The diastereometic isomers were separated by column chromatographywith ethyl acetate/hexane (2/8, v/v) as eluent to give RSS (0.2 g) andRRR (0.34 g) in 51% overall yield.

Compound 55: Compound 54 (4.2 g) was dissolved in ethyl acetate (200ml). 4N HCl in dioxane (4.35 ml) was added dropwise while stirring. Awhite precipitate resulted. The ethyl acetate was removed under reducedpressure, and the resulting white solid (4.6 g, 100%) was dried invacuo.

Compound 56: Compound 55 (4.6 g) was dissolved in 95% ethanol (150 ml)in a hydrogenation flask. 10% Palladium on activated carbon (0.5 g) wasadded. The flask was pressurized with hydrogen to 50 psi, rocked at roomtemperature for 22 hours, by which time NMR spectroscopy indicated thatthe hydrogenolysis was complete. The Pd/C was removed by filtration. Thefiltrate was concentrated to give a white solid. The white solid wasdissolved in acetone/water (2/1, v/v, 150 ml). NaHCO₃ (9.7 g) was added,followed by Cbz-OSU (3.4 g). The reaction mixture was stirred at roomtemperature for 14 hours. Water (100 ml) was added. The acetone wasremoved under reduced pressure. The aqueous mixture was extracted withethyl acetate (3×200 ml). The extracts were washed with 1N HCl (3×100ml) and saturated NaHCO₃ (aqueous), dried over MgSO₄, and concentratedto give a colorless oil. The crude product was purified by columnchromatography with ethyl acetate/hexane (3/7, v/v) as eluent lo givethe clean product as a colorless sticky oil (4.0 g) in 90% yield.

Compound 57: Compound 56 (2.0 g) was dissolved in methanol/water (3/1,v/v, 115 ml), cooled to 0° C., LiOH.H₂O (2.4 g) was added. The mixturewas stirred at 0° C. for 15 hours, by which time TLC indicated that thehydrolysis was complete. Saturated ammonium hydroxide (aqueous, 100 ml)was added. The methanol was removed under reduced pressure. The aqueouswas acidified with 1N HCl to pH 3, extracted with ethyl acetate (3×200ml). The extracts were washed with dilute brine (100 ml), dried overMgSO₄, concentrated to give a foamy solid (1.63 g, 88%), which was useddirectly without further purification).

Compound 58: Compound 57 (1.63 g) was dissolved in methanol (70 ml) in ahydrogenation flask. 5% Palladium on activated carbon (250 mg) wasadded. The flask was pressurized with hydrogen to 35 psi, rocked at roomtemperature for 15 hours, by which time NMR spectroscopy indicated thatthe hydrogenolysis was complete. The Pd/C was removed by filtration. Thefiltrate was concentrated to give a white solid. The white solid wasdissolved in acetone/water (2/1, v/v, 90 ml), cooled to 0° C. NaHCO₃(2.27 g) was added, followed by FMOC-OSU (1.83 g). The reaction mixturewas stirred at 0° C. for 2 hours, then at room temperature for 28 hours.Water (50 ml) was added. The acetone was removed under reduced pressure.The aqueous was acidified with 1N HCl to pH 3, extracted with ethylacetate (3×200 ml). The extracts were washed with dilute brine (100 ml),dried over MgSO₄, concentrated to give a foamy white solid. The crudewhite solid was purified by column chromatography with methanolfethylacetate (3/7, v/v) as eluent to give the clean product as a white solid(1.68 g) in 84% yield.

The synthesis of this monomer is an extension of that given in Patel etal. (1997), J. Org. Chem. 62:6439.

N-tert-Butoxycarbonyl-L-Serine-β-Lactone 3.1. A solution oftriphenylphosphine (7.48 g, 28.5 mmol) in anhydrous THF (110 mL) wasstirred under N₂, cooled to −78° C., and dimethylazodicarboxylate (4.83mL, 30.7 mmol) was added dropwise. The mixture was stirred for 10 min,and a solution of Boc-serine (4.5 g, 21.9 mmol) in THY (110 mL) wasadded dropwise. After the addition, stirring was continued at −78° C.for 30 min, and for an additional 3 h after the cooling bath had beenremoved. The solution was concentrated, and the residue was purified bycolumn chromatography eluting with hexanes/ethyl acetate (2/1, v/v) toafford 2.21 g (54% yield) of the desired product as a white solid.

(S)-N²-(tert-Butoxycarbonyl)-N₃-(2-propenyl)-2,3-diaminopropanoic acid3.2. A N-tert-Butoxycarbonyl-L-serine-β-lactone (2.21 g, 11.8 mmol) inacetonitrile (224 mL) was added dropwise to a stirred solution ofallylamine (21.9 mL, 0.29 mmol) in acetonitrile (448 mL). The solutionwas stirred for 2 h at room temperature and then concentrated. The solidresidue was slurried with acetonitrile and filtered to afford 1.51 g(52% yield) of the desired product as a white solid.

(S)-N²-(tert-Butoxycarbonyl)-N³-(benzyloxycarbonyl)-N³-(2-propenyl)-2,3-diaminopropanoicacid 3.3. A solution of(S)-N²-(tert-Butoxycarbonyl)-N₃-(2-propenyl)-2,3-diaminopropanoic acid(2.80 g, 11.4 mmol) in saturated NaHCO₃ (36 mL) and H₂O (5 mL) wastreated dropwise with a solution of benzyl chloroformate (1.84 mL, 12.8mmol) in acetone (2.5 mL). The cloudy reaction mixture was stirred for 2h. The resulting solution was partitioned between diethyl ether (130 mL)and H₂O (65 mL). The aqueous layer was cooled in an ice bath, brought topH 2 with 1 M HCl, and extracted with ethyl acetate. The organicextracts were dried over MgSO₄ and concentrated to afford 3.15 g (73%yield) of the desired product as a colorless oil.

(S)-N¹-(tert-butoxycarbonyl)-N⁴-(benzyloxycarbonyl)-PiperazineCarboxylic Acid 3.5. A solution of(S)-N²-(tert-Butoxycarbonyl)-N³-(benzyloxycarbonyl)-N³-(2-propenyl)-2,3-diaminopropanoicacid (3.15 g, 8.3 mmol) in methylene chloride (110 mL) and methanol (11mL) was cooled to −78° C. under N₂. Ozone was passed through thesolution until a pale blue color persisted (6 psi O₂, 90 V, 20 min). Theexcess ozone was purged by bubbling N₂ through the solution for 15 min.Dimethyl sulfide (11 mL) was added, and the solution was allowed to warmgradually to room temperature overnight. After 20 h, the reactionmixture was diluted with methylene chloride (200 mL) and washed withbrine. The organic layer was dried over MgSO₄ and concentrated to afford3.02 g (95% yield) of the desired product as a yellow foam.

The crude material and triethylsilane (1.4 mL, 8.8 mmol) in methylenechloride (200 mL) under N₂ were cooled to −78° C. and treated dropwisewith boron trifluoride diethyl etherate (1.11 mL, 8.8 mmol). After 30min, more triethylsilane (1.4 mL, 8.8 mmol) and boron trifluoridediethyl etherate (1.11 mL, 8.8 mmol) were added in a similar fashion.The reaction mixture was stirred for 2 h at −78° C., brine was added,and the cold mixture was extracted with methylene chloride. The organicextracts were dried over MgSO₄ and concentrated. The crude product waspurified by column chromatography eluting with methylene chloride/ethylacetate/acetic acid (2/1/0.03, v/v/v) to afford 2.13 g (74% yield) ofthe desired product as a white solid.

(S)-N¹-(tert-butoxycarbonyl)-N⁴-(benzyloxycarbonyl)-Piperazine EthylEster 3.6.(S)-N¹-(tert-butoxycarbonyl)-N⁴-(benzyloxycarbonyl)-piperazinecarboxylic acid (4.66 g, 12.8 mmol) was dissolved in DMF (128 mL).Cs₂CO₃ (4.37 g, 13.4 mmol) and ethyl iodide (1.23 mL, 15.3 mmol) wereadded and the reaction mixture was stirred for 24 h at room temperature.The reaction mixture was concentrated, and the residue was dissolved inH₂O. The aqueous solution was extracted with ethyl acetate. The organiclayer was dried over MgSO₄ and concentrated. The crude product waspurified by column chromatography eluting with ethyl acetate/hexanes(1/1, v/v) to afford 3.77 g (75% yield) of the desired product as anoil.

(S)-N¹-(mesyl)-N⁴-(benzyloxycarbonyl)-Piperazine Ethyl Ester 3.7.(S)-N¹-(tert-butoxycarbonyl)-N⁴-(benzyloxycarbonyl)-Piperazine ethylester (3.77 g, 9.6 mmol) was dissolved in 4 N HCl/dioxane and stirredfor 2 h at room temperature. The reaction mixture was concentrated undera stream of N₂, then on the vacuum line. The residue was dissolved inmethylene chloride and cooled to 0° C. Triethylamine (6.7 mL, 50 mmol)and DMAP (0.12 g, 1.0 mmol) were added, followed by methanesulfonylchloride (1.5 mL, 19.2 mmol). The reaction solution was stirred for 24 hat room temperature. The reaction solution was then washed with brine,and the organic layer was dried over MgSO₄ and concentrated. The crudeproduct was purified by column chromatography eluting with ethylacetate/hexanes (1/1, v/v) to afford 2.1 g (59% yield) of the desiredproduct as an oil.

Boc-{(N⁴-Ts)-PiCA}₂-OBn 3.8. Via general procedure A,Boc-{(N⁴-Ts)-PiCA}-OBn (0.15 g, 0.3 mmol) was Boc-deprotected andcoupled with Boc-{(N⁴-Ts)-PiCA}-OH (0.12 g, 0.3 mmol). After workup, thecrude product was purified by column chromatography eluting with ethylacetate/hexanes (1/1, v/v) to afford 0.13 g (57% yield) of the desiredproduct as a colorless oil.

Boc-{(N⁴-Ts)-PiCA}₃-OBn 3.9. Via general procedure A,Boc-{(N⁴-Ts)-PiCA}₂-OBn (0.11 g, 0.2 mmol) was Boc-deprotected andcoupled with Boc-{(N⁴-Ts)-PiCA}-OH (58.3 mg, 0.2 mmol). After workup,the crude product was purified by column chromatography eluting withethyl acetate/hexanes (1/1, v/v) to afford 78 mg (51% yield) of thedesired product as a white foam.

Boc-{(N⁴-Ts)-PiCA}₄-OBn 3.10. Via general procedure A,Boc-{(N⁴-Ts)-PiCA}₃-OBn (65.2 mg, 0.1 mmol) was Boc-deprotected andcoupled with Boc-{(N⁴-Ts)-PiCA}-OH (24.9 mg, 0.1 mmol). After workup,the crude product was purified by column chromatography eluting withethyl acetate/hexanes (1/1, v/v) to afford 25 mg (33% yield) of thedesired product as a white foam.

Circular dichroism data for (N⁴-Ts)-PiCA oligomers in methanol (25° C.)suggest that the tetramer adopts a distinct secondary structure, whichis different than the structure adopted by the dimer and trimer.

cis-5-Methoxymethyl-3-Pyrrolidine Carboxylic Acid (cis-5-MOM-PCA)

trans-4Hydroxy-Cbz-L-Proline 4.1. Benzyl chloroformate (8.6 mL, 0.06mol) was dissolved in acetone (12 mL), and this solution was addeddropwise to a stirred solution of trans-4-Hydroxy-L-proline (6.56 g,0.05 mol) in satd. NaHCO₃ (160 mL) and H₂O (24 mL). The resultingsolution was stirred for 6 h at room temperature. The solution waswashed with diethyl ether, and the organic layer was discarded. Theaqueous layer was acidified with to pH 3 with 1 M HCl and extracted withethyl acetate. The organic layer was dried over MgSO₄ and concentratedto afford 13.5 g (quantitative yield) of the desired product as an oil.

trans-4-TBDMSO-Cbz-L-Proline 4.2. trans-4-Hydroxy-Cbz-L-proline (13.5 g,0.05 mol) was dissolved in DMF (190 mL), followed by the addition ofimidazole (17.0 g, 0.25 mol) and TBDMS-Cl (22.6 g, 0.15 mol). Theresulting solution was stirred for 12 h at room temperature. Methanol(150 mL) was added and the solution was stirred for 2 h. The solutionwas concentrated, the residue was dissolved in ethyl acetate and washedwith 1 M HCl. The organic layer was dried over MgSO₄ and concentrated.The crude product was purified by column chromatography eluting withmethylene chloride/ethyl acetate/acetic acid (2/1/0.03, v/v/v) to afford18.01 g (95% yield) of the desired product as an oil.

trans-5-Hydroxylmethyl-3-TBDMSO-Cbz-Pyrrolidine 4.3.trans-4-TBDMSO-Cbz-L-proline (14.31 g, 0.04 mol) was dissolved in THFand added via cannula to a stirred solution of 2 M Me₂S.BH₃ in THF (48.0mL, 0.09 mol). The resulting solution was stirred for 16 h at reflux.The reaction was then quenched with methanol (50 mL) and concentrated.The residued was dissolved in ethyl acetate and washed with brine. Theorganic layer was dried over MgSO₄ and concentrated. The crude productwas purified by column chromatography eluting with hexanes/ethyl acetate(3/1, v/v) to afford 9.73 g (70% yield) of the desired product as anoil.

trans-5-Methoxymethyl-3-TBDMSO-Cbz-Pyrrolidine 4.4.trans-2-Hydroxylmethyl-4-TBDMSO-Cbz-pyrrolidine (5.02 g, 13.7 mmol) wasdissolved in acetonitrile (13.7 mL), followed by the addition ofiodomethane (8.55 mL, 0.14 mol) and Ag₂O (6.36 g, 27.5 mmol). Theresulting reaction mixture was stirred for 12 h at reflux in the dark.The reaction mixture was then filtered through celite and the celite waswashed with acetonitrile. The filtrate was concentrated. The crudeproduct was purified by column chromatography eluting with hexanes/ethylacetate (3/1, v/v) to afford 4.05 g (78% yield) of the desired productas an oil.

trans-5-Methoxymethyl-3-Tosyl-Cbz-Pyrrolidine 4.5.trans-2-Methoxymethyl-4-TBDMSO-Cbz-pyrrolidine (8.99 g, 23.6 mmol) wasdissolved in THF (237 mL), followed by the addition of 1 M TBAF in THF(23.7 mL, 23.7 mmol). The resulting solution was stirred for 3 h at roomtemperature. The reaction was quenched with satd. NH₄Cl. The solutionwas concentrated, the residue dissolved in ethyl acetate and washed withbrine. The organic layer was dried over MgSO₄ and concentrated. Theresidue was dissolved in methylene chloride (230 mL) and cooled to 0° C.DMAP (3.37 g, 27.6 mmol) and triethylamine (7.7 mL, 66.2 mmol) wereadded, followed by p-toluenesulfonyl chloride (5.26 g, 27.6 mmol). Thereaction solution was stirred for 12 h at room temperature. The solutionwas washed with brine and the organic layer was dried over MgSO₄ andconcentrated. The crude product was purified by column chromatographyeluting with hexanes/ethyl acetate (2/1, v/v) to afford 8.67 g (90%yield) of the desired product as an oil.

cis-5-Methoxymethyl-3-Cyano-Cbz-Pyrrolidine 4.6.trans-5-Methoxymethyl-3-tosyl-Cbz-pyrrolidine (3.55 g, 8.8 mmol) wasdissolved in DMSO (8.8 mL). Finely ground NaCN(0.65 g, 13.2 mmol) wasadded, and the resulting reaction mixture was stirred 4 h at 80° C. Thesolution was cooled to room temperature, diluted with H₂O (9 mL) andbrine (9 mL), and extracted with ethyl acetate. The organic extractswere dried over MgSO₄ and concentrated. The crude product was purifiedby column chromatography eluting with hexanes/ethyl acetate (2/1, v/v)to afford 2.09 g (87% yield) of the desired product as an oil.

cis-5-Methoxymethyl-Boc-3-Pyrrolidine Carboxylic Acid{cis-Boc-(5-MOM)-PCA-OH} 4.7.cis-5-Methoxymethyl-3-cyano-Cbz-pyrrolidine (1.71 g, 6.2 mmol) wasdissolved in concentrated HCl and stirred for 12 h at 50° C. Thesolution was cooled to room temperature and neutralized with NaHCO₃. Thesolution was concentrated, and the residue was dissolved in methanol (62mL). Triethylamine (2.6 mL, 18.7 mmol) and Boc₂O (1.63 g, 7.5 mmol) wereadded, and the solution was stirred 12 h at 50° C. The solution wasconcentrated and the residue was dissolved in H₂O. The aqueous solutionwas washed with diethyl ether, and the organic layer was discarded. Theaqueous layer was acidified with to pH 3 with 1 M HCl, and extractedwith ethyl acetate. The organic layer was dried over MgSO₄ andconcentrated to afford 1.40 g (86% yield) of the desired product as anoil.

cis-5-Methoxymethyl-Boc-3-Pyrrolidine Benzyl Ester Acid{cis-Boc-(5-MOM)-PCA-OBn} 4.8. cis-5-Methoxymethyl-Boc-3-pyrrolidinecarboxylic acid (1.4 g, 5.3 mmol) was dissolved in DMF (26.5 mL). Cs₂CO₃(1.73 g, 5.3 mmol) and benzyl bromide (0.76 mL, 6.4 mmol) were added,and the reaction mixture was stirred 24 h at room temperature. Thereaction mixture was concentrated, and the residue was dissolved in H₂O.The aqueous solution was extracted with ethyl acetate. The organic layerwas dried over MgSO₄ and concentrated. The crude product was purified bycolumn chromatography eluting with ethyl acetate/hexanes (1/1, v/v) toafford 1.88 g (85% yield) of the desired product as an oil.

Boc-{(cis-5-MOM)-PCA}_(2-OBn) 4.9. Via general procedure B,cis-Boc-(5-MOM)-PCA-OBn (1.88 g, 5.38 mmol) was Boc-deprotected andcoupled with cis-Boc-(5-MOM)-PCA-OH (1.40 g, 5.38 mmol). After workup,the crude product was purified by column chromatography eluting withethyl acetate/hexanes (3/1, v/v) to afford 1.90 g (72% yield) of thedesired product as an oil.

Boc-{(cis-5-MOM)-PCA}₃-OBn 4.10. Via general procedure B,cis-Boc-{(5-MOM)-PCA}₂-OBn (0.26 g, 0.54 mmol) was Boc-deprotected andcoupled with cis-Boc-(5-MOM)-PCA-OH (0.13 g, 0.54 mmol). After workup,the crude product was purified by column chromatography eluting withethyl acetayte/methanol (20/1, v/v) to afford 0.25 g (75% yield) of thedesired product as a white foam.

Boc-{(cis-5-MOM)-PCA}₄-OBn 4.11. Via general procedure B,cis-Boc-{(5-MOM)-PCA}₂-OBn (0.26 g, 0.54 mmol) was Boc-deprotected andcoupled with cis-Boc-{(5-MOM)-PCA}₂—OH (0.20 g, 0.54 mmol). Afterworkup, the crude product was purified by column chromatography elutingwith ethyl acetate/methanol (15/1, v/v) to afford 0.24 g (57% yield) ofthe desired product as a white foam.

Boc-{(cis-5-MOM)-PCA}₅-OBn 4.12. Via general procedure B,cis-Boc-{(5-MOM)-PCA}₃-OBn (0.12 g, 0.18 mmol) was Boc-deprotected andcoupled with cis-Boc-{(5-MOM)-PCA}₂—OH (0.09 g, 0.20 mmol). Afterworkup, the crude product was purified by column chromatography elutingwith methylene chloride/methanol (10/1, v/v) to afford 0.12 g (83%yield) of the desired product as a white foam.

Boc-{(cis-5-MOM)-PCA}₆-OBn 4.13. Via general procedure B,cis-Boc-{(5-MOM)-PCA}₄-OBn (0.13 g, 0.17 mmol) was Boc-deprotected andcoupled with cis-Boc-{(5-MOM)-PCA}₂—OH (0.07 g, 0.17 mmol). Afterworkup, the crude product was purified by column chromatography elutingwith methylene chloride/methanol (10/1, v/v) to afford 70 mg (52% yield)of the desired product as a glassy solid.

Boc-(cis-5-MOM)-PCA-NMe₂ 4.14. Via general procedure B,cis-Boc-{(5-MOM)-PCA}₄-OBn (0.11 g, 0.41 mmol) was Boc-deprotected andcoupled with dimethylamine hydrochloride (0.04 g, 0.49 mmol). Afterworkup, the crude product was purified by column chromatography elutingwith ethyl acetate/hexanes (3/1, v/v) to afford 57 mg (50% yield) of thedesired product as an oil.

CD spectra (25° C., methanol) for the oligomeric series from the monomerto the hexamer of cis-5-MOM)-PCA are shown in FIG. 3. The CD dataindicate similar behavior to that described above for the Nip and PCAoligomer series: the “per residue”CD” shows a steady change from monomerto tetramer, and is essentially constant thereafter. As noted above,this appears to suggest that the secondary structure is maximized at thetetramer length.

The CD spectra shown in FIG. 4 compares the pentamers of the PCA, Nip,and cis-5-MOM-PCA series of compounds. Interestingly, the cis-5-MOM-PCApentamer CD curve is intermediate between the other two in terms of theminimum around 212 nm. This is slighly higher than the Nip pentamer andslightly lower than the PCA pentamer. While a detailed structuralconclusion cannot be drawn from these data, they do suggest that allthree pentamers may have related conformations.

Molecular Modeling Studies:

Computer Simulations of a 3₁-Helix of trans-3-carboxy-4methylpiperidine:

Because an oligomer of trans-3-carboxy-4-methylpiperidine (TCMP) cancontain no intramolecular hydrogen bonds, a regular helical structuremust be stabilized through intrinsic molecular preferences.

Thus, these conformational preferences must be determined before anyhelical structure can be evaluated. The three molecules shown below wereconstructed to determine the effect an alkyl substituent has on therotation of the C2-C3-C(O)-N1′ torsion. Each of these molecules was putthrough a dihedral drive simulation in MacroModel 6.0, using the AMBER*Cforce field and CHCl₃ GB/SA continuous solvation. See Mohamadi, F.,Richards, N. G. J., Guida, W. C., Liskamp, R., Lipton, M., Caufield, C.,Chang, G., Hendrickson, T., Still, W. C. J. Comput. Chem. 1990, 11, 440.(MacroModel—an Integrated Software System for Modeling Organic andBioorganic Molecules Using Molecular Mechanics); Christianson, L. A.Thesis. University of Wisconsin-Madison, 1997; and Still, Tempczyk,Hawley and Hendrickson, J. Am. Chem. Soc. 1990, 112, 6127, respectively.For each of the simulations, the desired torsion was rotated from0°-360° in 10° increments and minimized 1000 iterations after eachrotation. Also, the internal amide bond was constrained to 0° (or cis).

The relative energies were compared at each increment for all threemolecules, as shown in the graphs presented in FIGS. 2A and 2B, and thepreferred geometry around the C2-C3-C(O)-N1′ torsion determined. Thedifferences between molecules 2 and 4 are relatively minor; both havethree minima at approximately the same energies. The lowest-energyminimum for both occurs at a C2-C3-C(O)-N1′ torsion angle of 20°, andthe next minimum occurs at 90° with a relative energy of +1.0 kca/molfor 2 and +1.2 kcal/mol for 4. The final minimum is over 5.7 kcal/molhigher in energy for each molecule and occurs at 250°.

The differences between molecule 3 and the others, however, are morepronounced. First, the lowest-energy minimum occurs at a C2-C3-C(O)-N1′torsion angle of 90°; the next minimum is only 0.3 kcal/mol higher inenergy and occurs at 40°. The final minimum at 250° is slightly lower inenergy than for the others at +4.5 kcal/mol.

Once the conformational preferences are established, the stability ofhelices built from each molecule can be evaluated through dynamicssimulations. First, the unsubstituted monomer, nipecotic acid, wasstudied. A decamer of nipecotic acid was constructed residue by residue,minimizing after each addition, thereby creating a 5₁-helix. Theresulting helix was subjected to a 200 ps molecular dynamics simulationwith a timestep of 0.5 fs, again using AMBER*C and GB/SA continuousCHCl₃ solvation. The simulation was inconclusive.

The next helix evaluated was a decamer of TCMP. This helix wasconstructed by constraining the C2-C3-C(O)-N1′ torsion angle to the“ideal” geometry of 90° determined in the earlier dihedral drivesimulations. The minimized conformation is a compact 3₁-helix. A 200 psmolecular dynamics simulation was run for this decamer under the sameconditions as for the nipecotic acid decamer. Unlike the previoussimulation, this oligomer remained helical throughout the simulation,indicating a stable conformation. The helix also held up in a 200 pssimulation of harsher mixed-mode Monte Carlo/stoichastic dynamics.However, the most telling evidence for the stability of this helix comesfrom simulated annealing calculations.

The 3₁-helix of TCMP was subjected to two simulated annealingcalculations: one with a starting structure of a 3₁-helix and onestarting from a 5₁-helix, similar to the decamer of nipecotic acid. Bothsimulations consisted of a 50 ps segment of molecular dynamics (1 fstimestep) at 600K to disrupt the initial conformation and a coolingphase of 400 ps in which the temperature slowly dropped from 600K to50K. Once again, the AMBER*C force field and GB/SA continuous CHCl₃solvation were used. The methyl substituent was constrained to 70°±20°with a force constant of 1000 kJ/mol (239 kcal/mol) to prevent the ringfrom sticking in an unproductive diaxial conformation during the coolingphase, which is a known problem with simulated annealing on six-memberedrings. The final conformation from both simulations was mostly3₁-helical. Neither structure is a perfect helix though; both contain aring that is in a twist-boat conformation, introducing aberrations inthe helix. These results indicate that an oligomer of TCMP will form astable helical structure.

Similar results are found for modelling studies of oligomers oftrans-5-carboxy-2-methylpiperidine.

Combinatorial Chemistry:

The defined conformation conferred by the preferred polypeptidesdescribed herein makes these polyamide compounds highly useful forconstructing large libraries of potentially useful compounds viacombinatorial chemistry. Combinatorial exploration of functionalizedoligomers of the subject compounds has a potential yield of literallymillions of novel polypeptide molecules, all of which display awell-defined secondary structure.

The amino acids which comprise the finished peptides can befunctionalized prior to being incorporated into a polypeptide, orunfunctionalized polypeptides can be constructed and then the entireoligomer functionalized. Neither method is preferred over the other asthey are complementary depending upon the types of compounds which aredesired.

Combinatorial libraries utilizing the present compounds may beconstructed using any means now known to the art or developed in thefuture. The preferred methods, however, are the “split and pool” methodusing solid-phase polypeptide synthesis on inert solid substrates andparallel synthesis, also referred to as multipin synthesis.

The “split and pool” concept is based on the fact that combinatorialbead libraries contain single beads which display only one type ofcompound, although there may be up to 10¹³ copies of the same compoundon a single 100 μm diameter bead. The process proceeds as follows,utilizing standard solid-phase peptide synthesis protocols as describedabove:

Several suitable solid substrates are available commercially. Thesubstrates are generally small diameter beads, e.g. about 100 μm, formedfrom inert polymeric materials such as polyoxyethylene-graftedpolystyrene or polydimethylacrylamide. An illustrative substrate,marketed under the trademark “ARGOGEL” is available from ArgonautTechnologies, Washington, D.C.

Referring now to FIG. 5, which is a schematic depicting the split andpool method, a plurality of inert substrates are divided into two ormore groups and then a first set of subunits is covalently linked to theinert support. As depicted in FIG. 5, the initial plurality ofsubstrates is divided into three subgroups. The appearance of the threegroups of beads after the first round of coupling is shown at I of FIG.5. The three groups of beads are then pooled together to randomize thebeads. The beads are then again split into a number of subgroups.Another round of coupling then takes place wherein a second subunit isbonded to the first subunit already present on each bead. The process isthen repeated (theoretically ad infinitum) until the desired chainlength is attained.

The split and pool process is highly flexible and has the capability ofgenerating literally millions of different compounds which, in certainapplications, can be assayed for activity while still attached to theinert substrate.

A critical aspect of the split and pool methodology is that eachreaction be driven to completion prior to initiating a subsequent roundof coupling. So long as each coupling reaction is driven to completion,each substrate bead will only display a single compound. Because therate of reaction will differ from bead to bead as the libraryconstruction progresses, the beads can be monitored using conventionaldyes to ensure that coupling is completed prior to initiating anotherround of synthesis. The presence of only a single compound per beadcomes about because each individual bead encounters only one amino acidat each coupling cycle. So long as the coupling cycle is driven tocompletion, all available coupling sites on each bead will be reactedduring each cycle and therefore only one type of peptide will bedisplayed on each bead.

The resulting combinatorial library is comprised of a plurality of inertsubstrates, each having covalently linked thereto a differentpolypeptide. The polypeptides can be screened for activity while stillattached to the inert support, if so desired and feasible for theactivity being investigated. Beads which display the desired activityare then isolated and the polypeptide contained thereon characterized,e.g., by mass spectrometry. Where a solution-phase assay is to be usedto screen the library, the polypeptides are cleaved from the solidsubstrate and tested in solution.

As applied in the present invention, one or more of the subunits coupledto the inert substrate are selected from the cyclic imino acidsdescribed herein. In this fashion, large libraries of polypeptides canbe assembled.

An alternative approach to generating combinatorial libraries usesparallel synthesis. In this approach, a known set of first subunits iscovalently linked to a known location on a inert substrate, one subunittype to each location. The substrate may be a series of spots on asuitable divisible substrate such as filter paper or cotton. A substratecommonly used is an array of pins, each pin being manufactured from asuitable resin, described above.

After the initial round of coupling, each pin of the array bears a firstsubunit covalently linked thereto. The array is then reacted with aknown set of second subunits, generally different from the first,followed by reactions with a third set of subunits, and so on. Duringeach reiteration, each individual pin (or location) is coupled with aincoming subunit selected from a distinct set of subunits, with theorder of the subunits being recorded at each step. The final result isan array of polypeptides, with a different polypeptide bonded to eachsolid substrate. Because the ordering of the subunits is recorded, theidentity of the primary sequence of the polypeptide at any givenlocation on the substrate (i.e., any given pin) is known. As in thesplit and pool method, each coupling reaction must be driven tocompletion in order to ensure that each location on the substratecontains only a single type of polypeptide.

Large Molecule Interactions:

A use for the present compounds is as molecular probes to investigatethe interactions between biological macromolecules to identifyantagonists, agonists, and inhibitors of selected biological reactions.As noted above, many biological reactions take place between very largemacromolecules. The surface areas in which these reactions take placeare thought by many to be far too large to be disrupted, altered, ormimicked by a small molecule. It has been difficult, if not impossible,to manufacture molecular probes of modest size that display awell-defined conformation. Because the compounds described herein assumea highly predictable helical or sheet conformation, even whenfunctionalized, they find use as reagents to probe the interactionbetween large biomolecules.

Employing the combinatorial methods described herein greatly expands themedicinal application of the compounds as vast libraries of compoundscan be screened for specific activities, such as inhibitory andantagonist activity in a selected biological reaction.

What is claimed is:
 1. A compound comprising formula: X—{A}_(n)—Ywherein n is an integer greater than 1; and each A, independent of everyother A, is selected from the group consisting of:

wherein R¹, R², and R⁸ are independently selected from the groupconsisting of hydrogen, hydroxy, linear or branched C₁-C₆-alkyl,monocyclic aryl, monocyclic aryl-C₁-C₆-alkyl, C₁-C₆-alkyloxy, andaryloxy; provided that in at least one “A” moiety, one of R¹, R², or R⁸is not hydrogen or C₁-C₆-alkyl; and wherein one of X or Y is hydrogen oran amino-terminal capping group selected from the group consisting offormyl, acetyl, tBoc, and Fmoc, and the other of X or Y is hydroxy or acarboxy-terminal capping group selected from the group consisting ofNH₂, NH(alkyl), and N(alkyl)₂.
 2. The compound of claim 1, wherein R¹,R², and R⁸ are independently selected from the group consisting ofhydrogen, hydroxy, and linear or branched C₁-C₆-alkyl.
 3. The compoundof claim 1, wherein R R¹, R², and R⁸ are independently selected from thegroup consisting of hydroxy, monocyclic aryl, monocyclicaryl-C₁-C₆-alkyl, C₁-C₆-allkyloxy, and aryloxy.
 4. A method of preparinga combinatorial library of oligomers or polymers of cyclic iminocarboxylic acids of formula: X—{A}_(n)—Y wherein n is an integer greaterthan 1; one of X or Y is hydrogen or an amino-terminal protecting groupselected from the group consisting of formyl, acetyl, tBoc, and Fmoc,and the other of X or Y is hydroxy or a carboxy-terminal protectinggroup selected from the group consisting of NH₂, NH(alkyl), andN(alkyl)₂; and each A, independent of every other A, is selected fromthe group consisting of:

the method comprising at least two successive iterations of: (a)covalently linking a first A subunit via its C terminus to a pluralityof separable solid substrates, the first A subunit selected from thegroup consisting of compounds of structure:

wherein R¹, R², and R⁸ are independently selected from the groupconsisting of hydrogen, hydroxy, linear or branched C₁-C₆-alkyl,monocyclic aryl, monocyclic aryl-C₁-C₆-alkyl, C₁-C₆-alkyloxy, andaryloxy; provided that in at least one A subunit, one of R¹, R², or R⁸is not hydrogen or C₁-C₆-alkyl; and (b) randomly dividing the pluralityof substrates into at least two sub-groups; and (c) deprotecting thefirst A subunits attached to the solid substrates of the at least twosub-groups; then (d) in separate, independent reactions, covalentlylinking to the first A subunit of each of the at least two sub-groups, asecond A subunit independently selected from the group listed in step(a); and then (e) combining the at least two sub-groups into a singleplurality; and then (f) repeating steps (b) through (e).